Expression profiles of I antigen, IGnT, and C/EBPα, and ChIP analysis in adult and cord CD34⁺, CD71⁺, and E-cultured cells. (A) Flow cytometry analysis for I antigen expression. Expressions of the cell-surface I antigens were analyzed using flow cytometry and detected with anti-I mAb (1:5 dilution). Open and shaded areas represent cells detected with anti-I mAb and FITC-conjugated secondary antibody, and with FITC-conjugated secondary antibody only, respectively. (B) Expressions of IGnT and C/EBPα transcripts. Real-time PCR was used to quantify the IGnT and C/EBPα transcripts in the cDNA samples. The quantities of IGnT (left panel) and C/EBPα (right panel) transcripts were normalized to that of the β-actin transcript. Data were obtained from 3 detections; standard deviations are shown. (C) Western blotting for C/EBPα. Nuclear fractions of the adult and cord CD34⁺, CD71⁺, and E-cultured cells were analyzed for the expression of C/EBPα protein. After stripping, the membranes were detected with anti–histone H1 antibody. Vertical lines have been inserted to indicate repositioned gel lanes. (D) ChIP analysis. For ChIP analysis, 2 × 10⁶ adult and 1 × 10⁶ cord cells were used (top and bottom panels, respectively). The chromatin DNAs immunoprecipitated with anti-C/EBPα antibody and input DNA controls were used for PCR amplification (indicated as IP and input, respectively). CD71⁺ cells were used for the no-antibody controls in PCR amplification (leftmost lanes).